The purpose of this research proposal is to acquire a better understanding of the molecular events occurring in the human platelet membrane during the transition of platelets from the resting to the activated, hemostatically-effective state. Membrane glycoproteins (GP), especially GP Ib, GP IIb and GP IIIa, are thought to act as receptors for platelet binding proteins, such as factor VIII/von Willebrand factor and fibrinogen, which are requisite cofactors of platelet-vessel wall interaction and platelet-platelet cohesion, respectively. Recent evidence suggests that the induction of fibrinogen receptor activity involves calcium-mediated conformational changes and/or reassociations of GP IIb and GP IIIa within the membrane. In order to ascertain the extent of complex formation in resting as opposed to activated platelets, and thus the role of complex formation in fibrinogen receptor induction, I propose to study the calcium-mediated interaction of GP IIb and IIIa in greater detail using site-specific murine monoclonal antibodies and immunoelectron microscopy, immunofluorescence microscopy and electron spin resonance techniques. Since recent evidence indicates that GP Ib exists in the membrane of intact platelets in a form that may be structurally and functionally different from that hitherto analyzed in vitro, similar methods will be applied to an analysis of GP Ib and its "structural analogues," including glycocalicin, with the membrane of intact platelets. The endogenous calcium-activated protease (CAP) of platelets is thought to modulate platelet activation by cleavage of certain cytoskeletal elements (actin-binding protein and P235) or glycoprotein Ib. Having recently obtained evidence suggesting that endogenous platelet fibrinogen and factor VIII/von Willebrand factor are also substrates of this protease, I propose to investigate the potential role of CAP in the modulation of platelet activation via cleavage of these proteins. These investigations should lead to a better understanding of the phenomena of platelet adhesion and platelet aggregation at a molecular level and thus provide new insight into the etiology of thrombotic disorders and other pathologic conditions in which primary hemostasis and platelet function are compromised.